Safety Biomarkers Holy Grail of Toxicology

Kate Marusina, Ph.D.

There is a great need to reduce the cost of developing new pharmaceuticals, now approaching $900 million per drug. About 75% of this cost is attributed to failed drugs. The FDA estimates that a 10% improvement in predicting clinical trial failures could reduce the average cost of drug development by nearly $100 million.

Using more discriminatory tests of efficacy and toxicity should eliminate suboptimal compounds earlier in development. Traditional preclinical animal studies often do not reflect human-specific metabolic and toxic effects. Most commonly used toxicity biomarkers appear only when a significant organ damage has occurred.

Toxicology’s holy grail remains finding translational and safety biomarkers that can predict or anticipate toxic manifestation and detect damage earlier in human trials. Several companies presented approaches to discovery of toxicity biomarkers at the 2006 IBC Conference and CHI Biomarker World Congress.

Renal Biomarker Discovery by Histomics

Histomics® is a method of discovering biomarkers with unique tissue specificity as demonstrated by immunohistochemistry. The detection of such biomarker in biofluids signals early tissue damage. Knowing the origin of the biomarker makes interpretation of clinical events much easier. Histologically defined biomarkers are directly linked with histopathology as a standard for toxicological grading.

Biotrin International (www.biotrin.com), which coined the term Histomics, has developed a panel of early biomarkers of kidney damage. Kidneys are a major target of drug toxicity, but most of the damage remains subclinical for a long time. Clinical presentation as detected by a drop in glomerular filtration rate comes later when significant injury to the renal tubules has already occurred.

“Our goal is to develop site-specific early toxicity biomarkers for the nephron,” stated Joe Keenan, Ph.D., sales and marketing manager. “We injected rats with toxins known to affect specific areas of kidneys, then collected urine samples, isolated proteins, and developed monoclonal antibodies. Several of them demonstrated excellent specificity in staining frozen kidney sections.”

Assay development for clusterin, a-GST (a-glutathione-S-transferase), GST Yb1, and RPA-1 (renal papillary antigen) is now being validated by the International Life Sciences Institute’s division of Health and Environmental Sciences (ILSI HESI) Biomarker Technical Committee. RPA-1 shows great promise for detection of renal papillary necrosis, a common form of nephrotoxicity for which there are currently no good biomarkers.

“a-GST is one of our first and most-studied biomarkers,” continued Dr. Keenan. “In addition to proximal tubular kidney cells it is also a major component of hepatocytes. a-GST is released with even slight cellular damage, and it is a much earlier and more sensitive indicator of hepatotoxicity than such common markers as ALT, AST, and LDH. Elevated levels of transaminases may reflect other processes, such as muscle injury and changes in body mass or diet, whereas serum GST levels are unaffected by this. Furthermore, a-GST levels return rapidly to normal after the cessation of hepatocyte injury.”

Protein Adducts and Autoantibodies

Metabolism of drugs often produces reactive metabolites (RMs) that nonspecifically interact with catalytic enzymes or other proteins. Resulting protein adducts may produce toxic reactions by inactivating critical enzymes or initiating adverse immune reactions. With the advent of LC-MS and MALDI/TOF MS technologies, it is possible to study these drug-protein adducts, which can lead to a better mechanistic understanding of the role of protein adducts in toxicity.

“Some adverse reactions may be attributed to RMs,” pointed out John Erve, Ph.D., principle research scientist, drug safety, and metabolism, Wyeth Research (www.wyeth.com). “For example, disulfiram, or Antabuse, is metabolized to an RM that inactivates aldehyde dehydrogenase, but this same RM may also lead to idiosyncratic liver damage in some people in doses that are safe for the general population, although further work is necessary to support this latter hypothesis.”

During research at Vanderbilt University Medical Center, Dr. Erve and coworkers developed a methodology for early detection of neurotoxicity, based on protein adducts. Industrial solvents, such as carbon disulfide and hexane, are known neurotoxins causing axonal swelling and disruption of axonal transport. Dr. Erve demonstrated that carbon disulfide is capable of crosslinking neurofilaments. Carbon disulfide also reacts with hemoglobin and spectrin and can be detected by MS analysis of blood samples.

“We can detect protein adducts in blood within two weeks after the exposure, whereas neurological damage is not diagnosed until thirteen weeks. Finding preneurotoxic biomarkers is important, because you can prevent further exposure before irreversible damage occurs,” added Dr. Erve. “If we learn how protein adducts cause damage, we may be able to better predict whether drugs with similar chemical structures would cause similar toxicities.”

In silico Pharmacogenomic Biomarker Discovery

Gene Logic (www.genelogic.com) created the ToxExpress® System, a database containing gene expression profiles from rat and human primary hepatocyte samples treated with various drugs. In addition, the company collects histopathological and physiological information. To date, the company has accumulated over 14,000 tissue samples and catalogued the effects of hundreds of different reference compounds.

Gene Logic’s BioExpress System contains gene expression data from over 20,000 normal and diseased human tissues along with their relevant clinical information. Gene Logic places a special emphasis on early data points—within hours of administering the compound—to enable customers to use similar time points to find classical toxicities as well as adverse events that may be overlooked during classical toxicity screening.

“Pharmaceutical companies want to find new translational biomarkers of liver damage,” said Donna L. Mendrick, Ph.D., scientific fellow and vp, toxicogenomics at Gene Logic. “Hepatotoxicity continues to be a major cause of drug failure and can result in withdrawal of a drug from clinical trials or from the market.” Approximately 75% of the idiosyncratic drug reactions result in liver transplantation or death. Liver biopsy is a diagnostic gold standard but it often comes too late to reverse the injury.

Gene Logic used its gene expression approach to find blood-based biomarkers that indicate subclinical liver damage in rats and may be able to predict liver damage in humans. By using statistical analysis of differential gene expression profiles of normal and diseased liver samples, Dr. Mendrick’s group found 80 genes that encode secreted proteins, including fetuins. Fetuin-B (FETUB) expression is highly specific for human liver, and it is down-regulated in fibrotic and malignant livers.

To determine whether FETUB could serve as a translational biomarker, a gene expression comparison between normal rat tissues and tissues treated with hepatotoxic compounds was performed. “We demonstrated that FETUB is down-regulated by compounds known to cause genotoxicity and fibrotic changes,” added Dr. Mendrick. “The next step would be biological validation and assay development.”

“Novel safety biomarkers still face huge resistance. Pharma shows great reluctance in adopting new biomarkers of drug safety, citing the lack of sufficient data on correlation with the adverse effects,” cautioned Rakesh Dixit, Ph.D, global head and senior director, toxicology at MedImmune(www.medimmune.com).

“Substantial clinical efforts will be required to attain the level of comfort associated with old biomarkers, such as creatinine and BUN, liver function tests, etc.

“An alternative route is to collect the data on new biomarkers concurrently with other clinical trial information. The same blood sample could be used for measurement of creatinine and for the levels of a new biomarker. If not for submission purposes, this data could be used to enrich our understanding of biomarker relationship to pathophysiological processes related to diseases and drug-induced adverse effects.”

However, it seems like the Big Pharma is catching onto the idea of adding a few new biomarkers to its portfolio and is willing to undertake data sharing and collaboration to an unprecedented extent. In 2003 several pharma companies funded the Consortium on Metabonomic Toxicology (COMET). The objective of the project was to investigate the utility of metabonomic approaches to the toxicological assessment of drug candidates. The consortium used 1H NMR spectroscopy to classify the biofluids in terms of known pathological effects caused by administration of known toxic substances.

COMET just completed a three-year effort, which resulted in a database of NMR spectra (biofluid fingerprints) covering 150 compounds. The database has already proven successful in tests of liver and kidney toxins. The second phase of the project, which aims to perform more mechanistic toxicology studies and supplement the current database with mass spectrometry data, is scheduled to begin in January.

Fifteen of the largest pharma firms are members of the ILSI HESI Technical Committee on the Development and Application of Biomarkers of Toxicity. The mission of the Committee is to develop a systematic approach for the evaluation of biomarkers that bridge preclinical and clinical stages of drug development and to provide and open forum regarding the application of these biomarkers in risk assessment.

Most notably, the FDA took a proactive stand with its Critical Path Initiative to “stimulate and facilitate a national effort to modernize the scientific process through which a potential human drug, biological product, or medical device is transformed from a discovery or proof-of-concept into a medical product.”

Through its Critical Path Opportunities List and Report the FDA placed a special emphasis on applying novel technologies to improve the accuracy of the predictive tests determining the safety and efficacy of investigational medical products. A particular importance is assigned to qualification and validation of biomarkers of early toxicity, especially for critical organs, such as liver, kidney, and heart.

The Critical Path Institute sprung from this initiative. “Critical Path is a novel concept. However, the FDA does not fully finance this enterprise and relies heavily on pharmaceutical company member fees to continue the research,” noted Dr. Dixit. “This may be a significant barrier to a small company, even with promising biomarkers. It is a very encouraging development, but we are yet to find out if big pharmaceutical companies are willing to share their data in a truly meaningful way.”